Lithium reverses the effect of opioids on eNOS/nitric oxide pathway in human umbilical vein endothelial cells

The main challenge of pain management with opioids is development of acute and chronic analgesic tolerance. Several studies on neuronal cells have focused on the molecular mechanisms involved in tolerance such as cyclic AMP (cAMP) activation, and nitric oxide (NO) pathway. However, the effects of opioids on non-neuronal cells and tolerance development have been poorly investigated. Lithium chloride is a glycogen synthase kinase 3β (GSK-3β) inhibitor and exert its effects through modulation of nitric oxide pathway. In this study we examined the effect of lithium on acute/chronic morphine and methadone administration in endothelial cells which express mu opioid receptors. Human umbilical vein endothelial cells (HUVECs) were treated with different doses of morphine, methadone, and lithium for six and 48 h. Then we evaluated cell viability, nitrite and cyclic AMP levels, as well as the expression of endothelial nitric oxide synthase (eNOS) protein using Immunocytochemistry (ICC) assay and phosphorylated GSK-3β enzyme by western blot analysis in cells. Both chronic morphine and methadone treatment increased NO level and eNOS expression in HUVECs. Morphine induced cAMP overproduction after 48 h exposure with cells. Lithium pretreatment (10 mM) in both morphine and methadone received groups significantly reduced nitrite and cAMP levels as well as eNOS expression as compared to the control. The decreased amount of phospho GSK-3β due to the opioid exposure was increased following lithium treatment. Tolerance like pattern may occur in non-neuronal cells with opioid receptors and this study clearly revealed the attenuation of morphine and methadone tolerance like behavior by lithium treatment in HUVECs.